Scientists Identify Thousands of Proteins Associated with
the Deadliest Form of Malaria

Two scientists at The Scripps Research Institute (TSRI) led
a collaborative effort involving 18 researchers at half a
dozen laboratories in the United States and Great Britain
to determine the "proteome" of the most deadly form of the
malaria pathogen Plasmodium falciparum.

This study, in the current issue of the journal Nature,
accompanies an article detailing the completion of a major
six-year $17.9-million genome-sequencing effort involving
185 researchers from the United Kingdom, the United States,
and Australia that sequenced the entire Plasmodium falciparum
genome.

"This is the first instance that I know of where these proteomics
studies have gone along side-by-side with the genome sequencing
project," says TSRI Cell Biology Professor John Yates, who
was the lead scientist involved in the proteomics effort,
which identified the proteins in the single-celled Plasmodium
that cause malaria.

These efforts will pay huge dividends in global healthcare
if even a few of the newly identified proteins lead to the
development of new malaria vaccinesand Yates and his
colleagues found a total of more than 2,400 proteins.

"We don't exactly know the function of well over half of
the proteins identifiedwe just know that they are there,"
says Laurence Florens, who is a research associate at TSRI
and the lead author of the study.

Malaria is a nasty and often fatal disease, which may lead
to kidney failure, seizures, permanent neurological damage,
coma, and death. There are four types of Plasmodium parasites
that cause the disease, of which falciparum
is the most deadly.

Knowing which proteins are expressed by Plasmodium falciparum
should help scientists understand how the pathogen causes
malaria and, with luck, how to thwart it. That was the goal
of the proteomics approach taken by Florens and Yates.

Where "genomics" maps the DNA sequence and genes in an organism
like Plasmodium falciparum, "proteomics" adds the topographical
information to that map by identifying which genes are actually
expressed as proteins in the Plasmodium falciparum
cells.

More importantly, Florens and Yates also sought to identify
which proteins are expressed at which stages of the organism's
lifecycle. This was no small task. Plasmodium falciparum
has at least ten distinct stages in its lifecycle, and there
is no way of telling which are expressed at each distinct
stage of the pathogen's lifecycle simply by looking at the
genes.

But Florens and Yates were able to figure out which proteins
were expressed during four different stages (sporozoites,
merozoites, trophozoites, and gametocytes) and, thus, which
might make good vaccine targets.

Mass Spectrometry and Malaria

The process was basically to take samples of a single isolate
of Plasmodium falciparum and grow three of the four
different stages in blood in a way that allowed samples to
be purified. The fourth stage, the sporozoites, had to be
hand-dissected from mosquito salivary glands.

In purifying the samples, Yates and Florens first separated
the soluble proteins from the membrane-bound proteins, then
digested them (chopped into smaller "peptide" pieces with
enzymes), and resolved them using liquid chromatography combined
with tandem mass spectrometry.

The instrument detects the pieces and uses sophisticated
software that Yates and his colleagues developed previously
to search a database of predicted genes to reconstruct most
of the proteins in the sample. This technique was particularly
useful in this context because it allowed a very large background
"noise" of mosquito and human proteins to be subtracted out.
The peptides that come from the Plasmodium can be distinguished
from those that come from the mosquito or the human.

Furthermore, using the technique, Florens and Yates were
able to show not only which genes were expressed in each stage
of the Plasmodium falciparum life cycle, but which
proteins were membrane-associated, and which were inside the
cellimportant pieces of information for vaccine design.

One unexpected finding was that a lot of the proteins that
were expressed in particular stages "co-localized" in chromosomal
gene clusters possibly under the control of common regulatory
elements.

Promoters are regions of DNA in front of a gene that "turn
on" that gene like a switch and cause it to be expressed as
protein. Normally, any given gene will have its own promoter.
But Florens and Yates found many different clusters of genes
that become expressed together and might be under the control
of a single promoter. Florens and Yates believe that this
is one of the ways that the pathogen is able to thrive in
two different organisms (mosquitoes and humans).

"The switching between stages is something that happens
very fast," says Florens, "and [the pathogen] needs a mechanism
to express many genes quickly."

This work was supported by the Office of Naval Research,
the U.S. Army Medical Research and Material Command, and the
National Institutes of Health.

The authors of the paper are affiliated with the following
institutions: The Scripps Research Institute; Syngenta Research
& Technology; the Imperial College of Science, Technology
& Medicine; the Naval Medical Research Center; the National
Institute for Medical Research; the American Type Culture
Collection; and The Institute for Genomic Research.